Electrophoresis in Sieving Media
Electrophoretic sieving media are used to size separate biopolymers: nucleic acids, polysaccharides, and proteins. They provide a system of obstacles (typically gel or entangled polymers) in the electrophoretic migration path so that the migrating biopolymers collide with the obstacles and these collisions suppress their apparent migration velocity. (The first electrophoretic sieving media were starch and polyacrylamide gel.) The size separation is based on the fact that the electrophoretic migration of larger molecules and particles is retarded more than that of small molecules. Nucleic acids are equally ionized at non-acidic pH and have sufficient charge and mobility. They need not be modified to size separate during electrophoretic migration in sieving media. On the other hand, protein ionization and charge significantly vary depending on the amino acid composition. Therefore, native proteins are not size separated in sieving media in the absence of ionic surfactants. However, when heated with an ionic surfactant, proteins denature and bind the ionic surfactant, generating complexes with more or less equal surface charge density. These complexes migrate in sieving media according to their size.
Slab Gel Electrophoresis
SDS electrophoresis in polyacrylamide slab gel (SDS PAGE) was the first method separating proteins according to their size1-4. Shortly after the invention of SDS PAGE, a method separating proteins by polyacrylamide gel electrophoresis (PAGE) in the presence of cationic surfactants was described5. A study observing the migration behavior of protein-cationic-surfactant-complexes followed, predicting a failure of the electrophoresis in the presence of cationic surfactants to determine molecular weights of proteins6. Later, cetylpyridinium chloride7 and cetyltrimethylammonium bromide8-12 were used for size separations of proteins by PAGE. Several protocols have been developed to denature proteins with cetyltrimethylammonium bromide8-12.
Capillary Electrophoresis
When electrophoresis of proteins in sieving media was transferred from slab gels into capillaries, crosslinked polyacrylamide gel was initially used as a sieving matrix13,14. When linear hydrophilic polymers were introduced as a replaceable sieving matrix for separation of polynucleotides15, various polymers were utilized as a sieving matrix for electrophoretic size separation of biopolymers: linear polyacrylamide16-18, poly(ethylene oxide)19, dextran16, guaran20, glucomannan21, poly(vinyl alcohol)22, poly(hydroxypropyl acrylamide)23, poly(ethoxyethyl acrylamide)24, agarose25, and pullulan26. Size separations of proteins by capillary electrophoresis were performed mostly by SDS capillary sieving electrophoresis (CSE) in the molecular-weight range between about 14,000 and 205,000. The method was also modified for the size separation of proteins on microchip27 with poly(dimethyl acrylamide) as a sieving polymer28. Capillary electrophoresis meant a number of advantages as compared to electrophoresis in slab gel: faster analysis, automation, higher separation efficiency, and higher detection sensitivity. Nevertheless, a small size of capillaries emphasized the effect of the capillary wall: typically fused silica capillaries were used that contained ionized silanol groups on their internal surface, resulting in strong wall adsorption, significant electroosmotic flow, eddy migration, and consequent mediocre separation efficiency. Electroosmotic flow was eventually suppressed by applying a hydrolytically stable neutral coating on the capillary wall (U.S. Pat. No. 5,143,753). Nevertheless, in SDS CSE, SDS adsorbs on the neutral coating and generates secondary electroosmotic flow. Mediocre reproducibility and separation efficiency are the results of this deleterious effect. Currently, SDS CSE is performed in bare capillaries after extensive rinsing of the capillary between runs, significantly reducing the throughput of the analysis (U.S. Patent Application 20090314638). Hypothetically, electroosmotic flow in SDS CSE could be also suppressed by reducing pH of the sieving medium and a consequent suppression of the silanol ionization in the capillary wall. However, SDS binding of proteins is weaker at pH<6 and SDS electrophoresis at this pH results in significantly broader peaks29 excluding this alternative from a real world practice.
Anomalous Protein Migration
Anomalous migration of some proteins was observed already in the early years of SDS PAGE when lysozyme and ribonuclease A did not migrate as expected from their molecular weights1,4,5. The authors speculated the anomalous proteins did not completely unfold and/or were not saturated with SDS4. Later it was found that even a single substitution of a neutral amino acid in α-crystallins resulted in changed mobility in SDS and thus different molecular weight30. Electrostatic repulsion between SDS and strongly acidic proteins could have been the cause for lower SDS binding31. Also glycoproteins were proposed to bind SDS below its saturation since hydrophilic carbohydrates were not likely to strongly bind SDS. William and Gratzer hypothesized the anomalously slow migration of acidic ferredoxins in SDS PAGE was caused by insufficient surfactant binding due to electrostatic repulsion of SDS and protein carboxylic groups5. This idea was corroborated by an observation that some acidic proteins, such as pepsin, papain, and glucose oxidase did not bind measurable amount of SDS32. Similarly, maleylation of cyanogen bromide fragments of cytochrome c significantly reduced their apparent molecular weights while that of native cytochrome c was not significantly affected by carbamylation33. Lysozyme was also modified by a reaction with dithio-compounds with various charges4. Carboxyethyl-, hydroxyethyl-, and aminoethyl-lysozyme derivatives migrated more anodically than lysozyme itself in 8 M urea, in absence of any ionic surfactant. The mobility differences in SDS PAGE indicated the intrinsic charge had an effect on the amount of SDS bound to the proteins.
Guttman and Nolan investigated the accuracy of molecular weights of 65 proteins as determineded by SDS electrophoresis in capillary and slab gel format. Independently of the format, more than one fourth of proteins exhibited biased migration34.
Normalization of Biased Molecular Weights
It was suggested to use so called Ferguson plot to correct the molecular weight of proteins with biased migration35,36.
Based on the hypothesis that electrostatic repulsion between ionic surfactant and proteins cause biased migration in electrophoretic size separation, several methods have been tested to normalize protein migration. The anomalously slow migration of acidic ferredoxins in SDS PAGE was normalized by esterification of their carboxyl groups with methanols.
Deglycosylation of several glycoproteins with N-glycosidase F improved the accuracy of molecular weights of these glycoproteins in SDS PAGE and SDS CSE on microchip37.
Several reaction schemes have been used to modify proteins to detect them by laser fluorescence detection. Some of them can be used for sample preparation to normalize protein migration in electrophoresis in the presence of a ionic surfactant.
Modifying Proteins by Carbamylation of Amino Groups
Protein amino groups can be modified by carbamylation with cyanate when homocitrulline is formed38 CNO−+H2N—CH2—CH2—CH2—CH2—CH(—NH—CO—)→H2N—CO—NH—CH2—CH2—CH2—CH2—CH(—NH—CO—)Modifying Proteins on Carboxylic Groups by Reaction with EDC
Proteins can be modified on their carboxylic groups by a reaction with a water-soluble carbodiimide, e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, (CH3)2—N—(CH2)3—N═C═N—CH2—CH3). Unstable acylurea ester is formed first then it reacts with a primary amine38 (CH3)2—N—(CH2)3—N═C═N—CH2—CH3+R1COOH→R1COO—C(N—CH2—CH3)═N—(CH2)3—N(CH3)2 R1COO—C(N—CH2—CH3)═N—(CH2)3—N(CH3)2+R2NH2→R1CO—NH—R2 
The participation of a primary amine in the reaction also means the reaction can be hypothetically used to modify protein amino groups by a reaction with a carboxylic acid and EDC.
Modifying Proteins on Amino Groups by Reaction with Isothiocyanate Derivatives
The reaction of protein amino groups with isothiocyanate derivatives has been widely used to label proteins with a fluorescent dye for their laser-induced fluorescence detection in HPLC and other separation methods. Isothiocyanates react with primary amines forming thiourea derivatives39 R1N═C═S+Protein-NH2→RiNH—CS—NH-ProteinModifying Proteins on Amino Groups by a Reaction with Succinimidyl Ester Derivatives
The reaction of protein amino groups with succinimidyl ester derivatives has been also used for labeling proteins with a fluorescent dye. Succinimidyl ester reacts with primary amines and forms carboxamide derivatives39 R1CO—O—N(CO—CH2—CH2—CO)+R2NH2→R1CO—NH—R2+HON(CO—CH2—CH2—CO)Modifying Proteins on Amino Groups by a Reaction with Sulfonyl Chloride Derivatives
Another reaction of protein amino groups that is to label proteins is with sulfonyl chloride derivatives. Sulfonyl chlorides react with primary amines and form sulfonamide derivatives39 R1—SO2Cl+R2NH2→R1—SO2—NH—R2 Modifying Proteins on Amino Groups by a Reaction with Aldehyde Derivatives
The reaction of protein amino groups with aldehyde derivatives has been also used for labeling proteins with a fluorescent dye when a Schiff base is formed first and then it is reduced to a corresponding alkylamine39 R1CO—H+R2NH2→R1CH═N—R2+H2O→RiCH2—NH—R2 Modifying Proteins on Sulfhydryl Groups by a Reaction with Charged Dithio Derivatives
Proteins can be modified by reaction of their disulfide bridges with dithioderivatives in the presence of an excess thiol, where protein thiols are generatedR1SH+R2—S—S-Protein→R1—S—S—R2+HS-Protein
The protein thiols then react with disulfides generating proteins modified in their disulfides19 HS-Protein+R3—S—S—R3→R3—S—S-Protein+R3—SH
When the modifying disulfide carries a charged moiety the protein intrinsic charge can be also modified4.